NZ621481B2 - Anti-tie2 antibodies uses thereof - Google Patents

Abstract

Disclosed is an isolated antibody or antigen-binding fragment thereof that specifically binds human Tie2 and blocks the interaction between Tie2 and all four of Ang1, Ang2, Ang3 and Ang4, wherein the antibody or antigen-binding fragment interacts with amino acids 96-106 of SEQ ID NO:7, amino acids 139-152 of SEQ ID NO:7; and amino acids 166-175 of SEQ ID NO:7, as determined by hydrogen/deuterium exchange, and comprises the heavy and light chain complementarity determining regions (CDRs) of the antibody produced from a cell line deposited with the American Type Culture Collection under accession number PTA-12295. Also disclosed is the use of the above-described antibody or antigen-binding fragment thereof in the manufacture of a medicament for inhibiting the growth of a tumour in a patient. 39-152 of SEQ ID NO:7; and amino acids 166-175 of SEQ ID NO:7, as determined by hydrogen/deuterium exchange, and comprises the heavy and light chain complementarity determining regions (CDRs) of the antibody produced from a cell line deposited with the American Type Culture Collection under accession number PTA-12295. Also disclosed is the use of the above-described antibody or antigen-binding fragment thereof in the manufacture of a medicament for inhibiting the growth of a tumour in a patient.

Description

ANTI-TIEZ ANTIBODIES AND USES THEREOF FIELD OF THE ION The present invention s to antibodies, and antigen-binding fragments thereof, which are specific for human Tie2.

BACKGROUND Angiogenesis is the biologicai process whereby new blood vessels are formed.

Aberrant angiogenesis is ated with several disease ions including, e.g., proliferative retinopathies, rheumatoid arthritis and psoriasis. in addition, it is well estabiished that angiogenesis is critical for tumor growth and maintenance. Tie2 is a single transmembrane tyrosine kinase or that has been localized to the endotheliai cells of forming blood vessels and has been shown to play a role in angiogenesis. Tie2 ligands include the angiopoietins leg, Ang‘l. Ang2, AngB and Ang4). Blocking the interaction between Tie2 and one or more of its ligands is ed to have beneficial eutic effects in settings where it is advantageous to limit or block angiogenesis. {0003] Antibodies to Tie2 are mentioned, 62.9., in US Patent Nos. 6,365,154 and 6,376,653.

Nonetheless, there remains a need in the art for novel molecules capable of binding to Tie2. especially anti—Tie2 dies that can block the interaction of Tie2 with one or more Tie2 ligands such as AngZ, Such molecules would be useful for various therapeutic and diagnostic purposes.

BRIEF SUMMARY OF THE INVENTION {0004] The present invention provides antibodies that bind human Tie2. The antibodies of the invention are useful, inter site, for inhibiting Tie2-mediated signaling and for ng diseases and disorders caused by or related to Tie2 ty andfor signaling. According to n ments, the antibodies of the invention block the interaction between Tie2 and one or more Tie2 ligands such as Angi, AngZ, Ang3, andlor Ang4. {0005] The antibodies of the invention can be full—length (for example, an igGl or lgG4 antibody) or may comprise only an antigen-binding portion {for example, a Fab, 2 or son fragment), and may be modified to affect functionality. egg.: to eiiminate residual effector ons {Raddy et at” 2000‘ J. Immunoi. 1642l925—1933}. {0006] The present invention provides anti—Tie2 antibodies that have substantially the same binding characteristics as any of the exemplary anti-TieZ antibodies described herein. The present invention includes cell lines that produce the ie2 antibodies described herein. As non-limiting examples‘ cell lines which produce the exemplary antibodies H1M2055N and H2aMZTBON were deposited under terms in accordance with the st Treaty with the American Type Culture Collection (ATCC), 10801 University Blvd, Manassas, Va. 20110-2209 on er 2,2011. The deposited cell lines have been assigned the following accession numbers PTA-12295 (HIM2055N) and PTA-12296 (H2aM2760N) The present invention provides nucleic acid les encoding the exemplary anti-Tie2 antibodies described herein. Recombinant expression s carrying the nucleic acids of the invention, and host cells into which such vectors have been introduced, are also encompassed by the invention, as are methods of producing the antibodies by culturing the host cells under ions permitting production of the antibodies, and recovering the antibodies produced The present invention includes antibodies and antigen-binding fragments thereof comprising the he ary and light chain CDR amino acid sequences (HCDRl, HCDR2, HCDR3, LCDRl, LCDR2 and LCDR3) found within any of the exemplary anti-Tie2 antibodies bed herein, including the antibodies ed by the deposited cell lines PTA-12295 and PTA-12296. The t invention also includes antibodies and antigenbinding fragments thereof comprising the he ary and light chain variable domain amino acid sequences (HCVR and LCVR) found within any of the exemplary anti-Tie2 antibodies described herein, ing the dies produced by the deposited cell lines PTA-12295 and PTA-12296 The present invention includes any of the exemplary anti-Tie2 antibodies described herein having a modified 91ycosylation pattern. In some applications, modification to remove undesirable 91ycosylation sites may be useful, or an antibody lacking a fucose inoiety present on the oligosaccharide chain, for example, to increase antibody dependent cellular cytotoxicity (ADCC) on (see Shield et a1. (2002) JBC 277:26733). In other applications, modification of galactosylation can be made in order to modify complement dependent cytotoxicity (CDC) [0009a] According to one ment, there is provided an isolated antibody or antigen- binding fragment thereof that specifically binds human Tie2 and blocks the interaction between Tie2 and all four of An 91 , An 92, An 93 and An 94, n the antibody or antigen-binding nt interacts with amino acids 96-106 of SEQ ID N0:7, amino acids 139-152 of SEQ ID N0:7; and amino acids 166-175 of SEQ ID N0:7, as determined by hydrogen/deuterium eXchange, and comprises the heary and light chain complementarity determining regions (CDRs) of the antibody produced from a cell line deposited with the American Type Culture tion under accession number 295 t00.01 In another aspect, the invention provides pharmaceutical compositions comprising an anti-Tie2 antibody as described herein and a pharmaceuticalIy able carrier. In a related aspect, the invention features compositions which comprise a combination of an anti-Tie2 antibody and a second therapeutic agent. Exemplary agents that may be advantageously combined with an anti-Tie2 antibody include, without limitation, other agents that inhibit anti-Tie2 activity (including other antibodies or antigenbinding fragments thereof, e inhibitors, small molecule antagonists, etc. ) and/or agents which interfere with Tie2 am or downstream signaling.

In yet another aspect, the invention provides methods for inhibiting Tie2 ty using an anti-Tie2 dy or antigen-binding portion of an antibody of the invention, wherein the therapeutic methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising an antibody or antigen-binding fragment of an antibody of the invention. The disorder treated is any disease or ion which is improved, ameliorated, ted or ted by removal, inhibition or reduction of Tie2 activity. The anti-Tie2 antibody or antibody fragment of the invention may function to block the interaction between Tie2 and a Tie2 binding partner (e. g. , An 91 , An 92, An 93, and/or An 94), or otherwise inhibit the signaiing activity of Tie2. {0012] The present invention also es the use of an anti-Tie2 antibody or antigen binding n of an antibody of the invention in the manufacture of a medicament for the treatment of a e or disorder rotated to or caused by Tie2 activity in a patient. {0013] Other embodiments wiil become apparent from a review of the ensuing detaiied description.

BRIEF PTION OF THE S {0014] Fig. 1. Linear depiction of tuli-iength human Tie2 and various deietion constructs used to map the epitope of the anti—Tie? antibodies of the present invention. Numbers above the constructs indicate the amino acid boundaries of the constructs reiative to the full-length Tie2 le {SEQ iD N021). {0015] Fig. 2. Histogram g the extent of angiopoietin (hAngi, hAng2, mAngS or hAng4) binding to a hTie2-coated sensor tip ore-treated with anti-Tie2 antibody H4H2055N or Control I. {0016] Fig. 3. Histogram showing the extent of anti-TieZ antibody (H4H2055N or l i) binding to a hTieE—coated sensor tip ore—treated with 100 nM of angiopoietin {hAng1, hAng2, mAngS or hAng4}. {0017] Fig. 4. Panel A shows the H29 tumor growth curves in mice ing administration of anti—Tie2 antibody 5N {I} or F0 controi (a). Downward arrow indicates the treatment start date. Panel B shows the tumor growth from the start of treatment in mice treated with anti- Tie2 antibody H2M2055N or PC controi. Asterisk (*) tes p<0.05 Mann Whitney non- parametric two—taiied t-test. {0018] Fig. 5. H29 tumor vessel density measured at the end of the experiment in mice treated with anti-Tie2 antibody H2M2055N or Fc controi. Asterisk F) indicates p<0.05 Mann Whitney non-parametric two-tailed t—test. {0019] Fig. 6. Panel A shows the 8010305 tumor growth curves in mice following administration of ie2 dy H2M2055N (I) or Fc control (0). Downward arrow tes the treatment start date. Panei B shows the tumor growth from the start of treatment in mice treated with anti-Tie2 antibody H2M2055N or F0 controi. Asterisk (*) indicates p<0.05 Mann Whitney non—parametric two-tailed t—test. {0020] Fig. 7. Colo205 tumor vessei y measured at the end of the experiment in mice treated with anti-Tie2 antibody H2M2055N or F0 controi. Asterisk (*) indicates p<0.05 Mann Whitney non—parametric two—tailed t—test.

DETAILED DESCRIPTION {0021] Before the present invention is described. it is to be understood that this invention is not iimited to particuiar methods and experimentai conditions described, as such methods and conditions may vary. it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not ed to be limiting, since the scope of the t invention will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term "about, " when used in reference to a particular recited numerical value, means that the value may vary from the recited value by no more than I%. For example, as used herein, the expression "about 100" includes 99 and 101 and allvalues in between (e. g. , 99.1,992,993,99.4, etc. ).

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.

Definitions [0023a] Comprises/comprising and tical variations thereof when used in this specification are to be taken to specify the presence of stated es, integers, steps or components or groups thereof, but do not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

The expressions "Tie2" and "Tie2 fragment, " as used herein refer to the human Tie2 protein or fragment unless specified as being from a non-human species (e. g. , "mouse Tie2, " "mouse Tie2 fragment, " "monkey Tie2, " y Tie2 fragment, " etc. ). A "Tie2 fragment" is any portion of Tie2 having fewer amino acids than the full-length Tie2 le and which is capable of binding to a Tie21igand. Human Tie2 has the amino acid sequence set forth in SEQ ID NO:I. Amino acid ces of Tie2 molecules from man species (e. g. , mouse, monkey, rabbit, dog, pig, etc. ) are available from public sources such as GenBank (e. g. , GenBank accession numbers NP_0387/8.2 (mouse); 099207. I (rat); etc).

The term "Tie21igand, " as used herein, means a protein with which the Tie2 protein cts to transmit a biological signal in vivo. The term "Tie21igand " includes any of the angiopoietins, including, e. g. , An 91, An 92, An93 and/or An 94. The term "An91, " as used herein, means a protein sing the amino acid sequence of SEQ ID N0:15, or a portion thereof which is capable of interacting with Tie2. The term "An 92 " as used herein, means a protein comprising the amino acid ce of SEQ ID N0:16, or a portion thereof which is capable of interacting with Tie2. The term "Ang3, " as used herein, means a protein comprising the amino acid sequence of SEQ ID N0:17, or a portion thereof which is capable of interacting with Tie2. The term "An 94, " as used herein, means a protein sing the amino acid sequence as set forth in of SEQ ID N0:18, or a portion thereof which is capable of interacting with Tie2.

The term "antibody", as used herein, is intended to refer to immunoglobulin molecules comprising four polypeptide chains, two he ary (H) chains and two light (L) chains inter connected by disulfide bonds, as well as ers f (e. g. , 19M). Each heavy chain comprises a he ary chain variable region (abbreviated herein as HCVR or VH) and a he ary chain constant region. The heavy chain constant region comprises three s, CHI, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CLI ). The VH and VL regions can be further subdivided into regions of hypervariabiiity, termed comptementarity determining regions {CDRs}, interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four F'Rs, arranged from amino-terminus to carboxy-terminus in the foiiowing order: FR‘l, CDRi, FR2, CDRZ, FR3, CDR3, FR4. in different embodiments of the invention, the FRs of the anti- Tie2 antibody (or antigen—binding n f) may be identicai to the human germline sequences, or may be naturally or artificiaiiy modified. An amino acid consensus sequence may be defined based on a side-by-side anaiysis of two or more CDRs. {0027] The term “antibody,“ as used herein, aiso includes antigen-binding fragments of foil dy molecuies. The terms "antigen—binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, include any naturally occurring, enzymaticatly obtainabie, synthetic, or caiiy engineered poiypeptide or glycoprotein that specificaiiy binds an antigen to form a x. Antigen-binding nts of an antibody may be derived, e.g., from fuii antibody molecules using any suitable standard ques such as proteotytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionaiiy constant domains. Such DNA is known and/or is readily avaiiable from, e.g., oommerciat sources, DNA iibraries (inciuding, e.g., phage-antibody libraries). or can be synthesized. The DNA may be sequenced and manipuiated chemicaily or by using molecuiar biology techniques, for example, to arrange one or more variabie and/or constant s into a suitable configuration, or to introduce codons, create ne residues, modify, add or delete amino acids, etc. {0028] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) Ftab')2 fragments: (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv {scFv} motecules; (vi) dAb fragments; and (vii) minimat ition units consisting of the amino acid residues that mimic the hyperyariable region of an antibody {e.g., an isolated complementarity determining region (CUR) such as a CURB e), or a constrained FR3-CDR3—FR4 peptide. Other engineered moiecuies, such as domain—specific antibodies, singie domain antibodies, domain- deieted antibodies, chimeric antibodies. afted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies {9.9. monovaientnanobodies, bivaient dies, etc), small modutar immunopharmaceuticals (SMiPs), and shark variable igNAR domains, are aiso encompassed within the expression en-binding fragment,“ as used . {0029] An antigen-binding fragment of an antibody wiil typicaily comprise at ieast one variabie domain. The variable domain may be of any size or amino acid composition and wit! generally comprise at least one CDR which is adjacent to or in frame with one or more ork sequences. in antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL s may be situated relative to one another in any suitable arrangement. For example, the le region may be dimeric and contain VH-VH, VH-VL or VL-VL dimers.

Atternativeiy, the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain. {0030] in certain embodiments, an antigen—binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting. ary configurations of le and constant domains that may be found within an antigen— binding fragment of an antibody of the present ion include: (i) VH—CH‘l; {ii} VH-CHZ; (iii) VH- CHS; (iv) VH-CH‘i-CHZ (v) VH-CHl-CHZ—CHB; (vi) -CHS; (vii) VHr-CL; (viii) VL-CHt; {ix} VL—CHZ; (x) VL-CHS; (xi) VL-CH‘l—CHZ; (xii) VL-CH‘l—CHZ—CHB; (xiii) VL-CHZ-CH3; and (xiv) VL—CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and nt domains may be either directiy linked to one another or may be linked by a full or partial hinge or linker region. A hinge region may consist of at least 2 (eg, 5, 10, 15, 20, 40, 6C! or more) amino acids which result in a flexible or semi—flexible linkage between adjacent variable andior nt s in a single polypeptide molecule.

Moreover, an antigen—binding fragment of an antibody of the present invention may se a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another andfor with one or more monomeric VH or VL domain (e.g., by disulfide fl. {0031] As with full antibody molecules, antigen-binding nts may be monospecific or pecific (eg, bispecific). A multispecitic antigen-binding fragment of an antibody will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a ent epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, may be adapted for use in the context of an antigen—binding fragment of an antibody of the present invention using routine techniques available in the art. {0032] The antibodies of the present invention may function through ment-dependent cytotoxicity (CD8) or antibody—dependent cell-mediated cytotoxioity (ADCC). "Complement- dependent cytotoxicity" (CDC) refers to lysis of antigen-expressing cells by an antibody of the invention in the ce of complement. "Antibody—dependent cell—mediated cytotoxicity“ (ADCC) refers to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (FcRs) (9.9., Natural Killer {NK} celis, neutrophiis, and macrophages) recognize bound antibody on a target cell and thereby lead to lysis of the target cell. CDC and ADCC can be ed using assays that are weil known and available in the art. (See, 9.9., US. ,500,362 and 337, and Ciynes et at. {1998) Proc. Natl. Acad. Sci. (USA) 95:652-656}.

The constant region of an antibody is imooitant in the ability of an antibody to fix complement and mediate cell-dependent cytotoxicity. Thus, the isotype of an antibody may be selected on the basis of whether it is desirable for the antibody to mediate cytotoxicity. {0033] The term "human antibody“, as used herein, is intended to e antibodies having variable and constant regions derived from human germline immunogiobulin sequences. The human antibodies of the invention may e amino acid residues not encoded by human germiine giobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in Wire or by somatic mutation in vii/o}, for e in the CDRs and in particuiar CDRB. However, the term ”human antibody", as used herein, is not intended to e antibodies in which CDR ces derived from the ne of another mammaiian species, such as a mouse, have been grafted onto human framework sequences. {0034] The term "recombinant human antibody", as used herein. is intended to include aii human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host ceii (described further beiow), antibodies isolated from a recombinant, combinatorial human antibody library (described further below), antibodies isoiated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see e.g., Tayior et at. (1992) Nucl. Acids Res. 20:6287-6295) or antibodies prepared, expressed, created or isolated by any other means that es ng of human immunoglobulin gene sequences to other DNA sequences.

Such recombinant human antibodies have variable and constant regions derived from human germiine immunoglobulin sequences. in certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or. when an animal transgenic for human lg sequences is used, in Vii/O somatic mutagenesis} and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and reiated to human germline VH and VL sequences, may not naturaiiy exist within the human antibody germline repertoire in viva. {0035] Human antibodies can exist in two forms that are associated with hinge heterogeneity. in one form, an immunogiobuiin molecule ses a stable tour chain construct of approximateiy t50—160 kDa in which the dimers are held together by an interchain heavy chain disuli‘ide bond. in a second form, the dimers are not linked via chain disutfide bonds and a moiecule of about 75-80 kDa is formed composed of a covalently coupied light and heavy chain (half-antibody). These forms have been extremely difficult to separate, even after affinity purification.

The frequency of appearance of the second form in various intact igG isotypes is due to, but not timited to, structural differences ated with the hinge region isotype of the antibody. A single amino acid tution in the hinge region of the human lgG4 hinge can significantiy reduce the appearance of the second form (Angai et al. (1993) Moiecular logy 30:105) to levels typicaliy ed using a human igGi hinge. The instant invention encompasses antibodies having one or more mutations in the hinge, CH2 or CH3 region which may be desirable, for exampie, in production, to improve the yieid of the desired antibody form. {0037] An "isolated antibody," as used , means an antibody that has been identified and ted andior recovered from at least one component of its naturai environment. For exampie. an dy that has been separated or removed from at ieast one component of an organism, or from a tissue or cell in which the antibody naturally exists or is naturally produced, is an "isolated antibody" for purposes of the present invention. An isolated antibody also es an antibody in situ Within a recombinant cell. Isolated antibodies are antibodies that have been subjected to at least one purification or isolation step. According to certain embodiments, an isolated antibody may be substantially free of other ar material andlor chemicals.

The term "specifically binds," or the like, means that an antibody or antigen—binding fragment thereoic forms a complex with an antigen that is relatively stable under physiologic conditions. s for determining whether an antibody specifically binds to an antigen are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. For example, an antibody that "specifically binds“ human Tie2, as used in the context of the present ion, includes antibodies that bind human Tie2 or portion f with a KB of less than about 1000 nM, less than about 500 nM, less than about 300 nM, less than about 200 anl, less than about will nM, less than about 90 nM, less than about 80 anl, less than about 70 anl, less than about 60 nit/E, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM. less than about 10 anl, less than about 5 nM, less than about 4 nh’l, less than about 3 nlvi, less than about 2 nit/l, less than about 1 nM or less than about 0.5 anl, as measured in a e plasmon resonance assay. (See. e. g, Example 2, herein}. An isolated antibody that specifically binds human Tie2 may, however, have cross- reactivity to other antigens, such as TieZ molecules from other (non-human) species. {0039] A “neutralizing" or "blocking" antibody, as used herein, is intended to refer to an antibody whose binding to Tie2: (i) eres with the interaction between Tie2 or a TieZ fragment and a Tie2 ligand (e.g., an angiopoietin}, andlor (ii) results in inhibition of at least one biological function of Tie2. The inhibition caused by a Tie2 neutralizing or blocking antibody need not be complete so long as it is detectable using an appropriate assay. ary assays for detecting TlE2 inhibition are described herein. {0040] The anti-Tie2 dies disclosed herein may comprise one or more amino acid substitutions, insertions andfor deletions in the framework andior CDR regions of the heavy and light chain le domains as compared to the corresponding germiine sequences from which the antibodies were derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases. The present invention includes antibodies, and antigenbinding fragments thereof, which are d from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework r CDR regions are mutated to the corresponding residue{s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of r human ne sequence, or to a conservative amino acid tution of the corresponding germline residue(s} (such sequence changes are referred to herein collectively as "germline mutations”). A person of ordinary skill in the art, starting with the heavy and tight chain variable region sequences disciosed herein, can easily produce numerous antibodies and antigen-binding fragments which comprise one or more individuai germline mutations or combinations thereof. in certain embodiments, aii of the framework and/or CDR residues within the VH andfor VL domains are mutated back to the residues found in the original germline sequence from which the antibody was derived. in other embodiments, oniy certain residues are mutated back to the originai germiine sequence, 9.9., oniy the mutated residues found within the first 8 amino acids of FR‘l or within the test 8 amino acids of FR4, or only the mutated es found within CDR’i. CDRZ or CDRS. In other embodiments, one or more of the ork and/or CDR e{s) are mutated to the corresponding residue(s) of a different germline sequence (i.e., a germline sequence that is different from the ne sequence from which the dy was aliy derived).

Furthermore, the antibodies of the present invention may contain any combination of two or more germline mutations within the framework r CDR regions, 9.9L, wherein certain individuai residues are mutated to the corresponding residue of a particuiar germiine sequence white certain other residues that differ from the originai germline sequence are maintained or are mutated to the corresponding residue of a different germline sequence. Once obtained, antibodies and antigen—binding fragments that contain one or more germiine mutations can be easily tested for one or more desired property such as, improved g specificity, increased binding affinity, improved or enhanced antagonistic or agonistic bioiogicai properties {as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed within the present invention. {0041] The present invention also es anti-Tiez dies comprising variants having one or more conservative substitutions as compared with the HCVR, LCVR andfor CDR amino acid sequences found within the exemplary anti—TieZ antibodies disciosed herein. For exampie. the present invention inciudes anti—Tie2 antibodies having HCVR, LCVR, andfor CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. vative amino acid tutions reiative to any of the HCVR, LCVR, andfor CDR amino acid sequences of the anti-Tie2 antibodies disciosed herein. {0042] The term “surface piasmon nce“, as used herein, refers to an opticai phenomenon that aiiows for the analysis of real-time interactions by detection of alterations in protein concentrations within a sor matrix, for example using the BiAcoreTM system (Biacore Life Sciences on of GE Heaithcare, Piscataway, NJ). {0043] The term "K53 as used . is intended to refer to the equiiibrium dissociation constant of a particular dy-antigen interaction.

The term "epitope" refers to an antigenic inant that interacts with a specific antigen binding site in the variabie region of an antibody molecuie known as a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to ent areas on an antigen and may have different biologicai s. Epitopes may be either conformational or linear. A conformational epitope is produced by spatially osed amino acids from different segments of the linear polypeptide chain. A linear e is one ed by adjacent amino acid es in a polypeptide chain. in certain stance, an epitope may include es of saccharides, phosphoryi groups, or sulfonyl groups on the antigen. {0045] The term “substantial identity“ or "substantially identical," when referring to a nucleic acid or fragment f, tes that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 95%, and more preferably at least about 96%, 97%. 98% or 99% of the nucleotide bases, as measured by any tnown algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain instances, encode a polypeptide having the same or substantially r amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule. {0046] As applied to polypeptides, the term “substantial similarity" or "substantially similar" means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFlT using default gap weights, share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity. ably, residue positions which are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain {R group} with similar chemical properties (e.g., charge or hydrophobicity). ln general, a conservative amino acid substitution will not substantially change the functional properties of a protein. in cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent ce ty or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well—known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307—331. Examples of groups of amino acids that have side chains with similar al properties include (i) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; {2) aliphatic-hydroxyl side chains: serine and threonine; (3} amide-containing side : asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate, and (7} sulfur-containing side chains are cysteine and methionine. Preferred conservative amino acids substitution groups are: valihe—leucine—isoleucine, phenylalanine— tyrosine, lysine-arginine, alanine—valine, ate-aspartate, and asparagine-glutamine.

Alternatively, a vative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet at a}. (1992) Science 256: 1443-1445. A ately conservative" replacement is any change having a nonnegative value in the PAMBSO log— likelihood . {0047] Sequence similarity for polypeptides. which is also referred to as sequence identity, is typicaily measured using sequence analysis software. Protein is software matches simiiar sequences using es of simiiarity assigned to various substitutions, deletions and other modifications, inciuding conservative amino acid substitutions. For instance, GCG software contains programs such as Gap and Bestfit which can be used with default parameters to ine sequence homology or sequence identity between closely related polypeptides, such as homologous ptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., 606 Version 6.1. Polypeptide sequences also can be compared using PASTA using default or recommended parameters, a program in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTAS) provides alignments and percent sequence identity of the regions of the best ovieriap between the query and search sequences (Pearson (2000) supra}. r preferred algorithm when comparing a sequence of the invention to a database containing a iarge number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN. using t parameters. See, 9.9L, Altschui et at. (1990} J. Moi. Bibi. 3410 and Aitschul at at. {1997) Nucleic Acids Res. 253389-402.

Bioiogical Characteristics of the Antibodies {0048] The present invention includes antibodies that block the interaction between Tie2 and a tie? ligand. As used herein, the expression ”blocks the interaction between Tie2 and 3 Tie? ligand" means that, in an assay in which the physical interaction between Tie2 and a Tie2 ligand can be detected and/or quantified, the addition of an antibody of the ion reduces the interaction n Tie2 and the Tie2 ligand (9.92, Ang1, Ang2, AngS andior Angfii) by at least 50%. A non-iimiting, exemplary assay that can be used to determine if an antibody blocks the interaction between human Tie2 and a Tie2 ligand is illustrated in Example 5, herein. in one exempiary embodiment of this assay format. dies are mixed with Tie2 protein, and then the antibody/Tie2 mixture is applied to a surface coated with a Tie 2 iigahd {in this case, AngE protein). After washing away d molecules, the amount of Tie2 bound to the Ang2— coated e is measured. By using varying amounts of antibody in this assay format, the amount of antibody required to biock 50% of Tie2 binding to Ang2 can be ated and expressed as an i059 value. The format of this assay can be reversed such that Tie2 is coated to e, antibody is added to the Tie2—coated e, unbound antibody is washed away, and then a Tie2 ligand is added to the dy—treated Tie2 surface. The present invention includes anti-TieZ antibodies that exhibit an leo of fess than about 100‘ nM when tested in a Tie2i'l'ie2 ligand binding assay as illustrated in Example 5, or a substantially similar assay. For exampie, the invention includes anti-Tie2 antibodies that exhibit an I055 of less than about 100, 90, 80, 70, 60, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, ‘l, 0.5, 0.4, 0.3, or 0.2 nM when tested in a Tie2fTie2 ligand binding assay as iilustrated in Exampie 5, or a substantially similar assay. r assay format that can be used to determine r an antibody blocks the interaction between human Tie2 and a Tie2 ligand is illustrated in Exampie 6, herein. in this assay format, a cell tine is used which is engineered to express human Tie2 on its surface, and which also includes a reporter construct that causes a able signal to be expressed when Tie2 interacts with a Tie2 iigand. The engineered coils are treated with anti—Tie2 dies and with Tie2 ligand and the reporter signal is measured. By using varying amounts of antibody in this assay format, the amount of antibody required to inhibit 50% of the reporter signal observed in the absence of antibody can be caiculated and expressed as an [C55 value. The present invention includes anti-Tie2 dies that exhibit an 1059 of less than about 20 nM when tested in a Tie2/Tie2 iigand binding assay as illustrated in Exampie 6, or a ntially similar assay.

For example, the ion includes anti—Tie2 antibodies that exhibit an “350 of less than about ,19,18,17,16,15,14,33,32,tt,10,9,8,7,5,5,4,3,2,1,0.5,0.4,0.3,0.2 or 0.1 MW when tested in a TierTieZ ligand binding assay as described in Example 6, or a substantiaily simitar assay.

Epitope Mapping and d Technologies The human Tie2 protein contains the following domains: an lgi domain, an igZ , an EGF repeat domain, an igS domain, and a fibronectin repeat (FN) domain ding FN’t, FN2 and FNS). These domains are graphicaily depicted in Figuret. The present ion includes anti-Tie2 antibodies which bind specificaily to an epitope within one or more of the foiiowing regions: (a) the 2—EGF domains {SEQ lD N027); (b) the lg2—EGF domains (SEQ lD N03); (0) the EGF domain {SEQ 1D N019); {d} the lg3-FN domains {SEQ ID N010); and/or (e) the FN domains {SEQ lD N011). (See Exampies 3 and 4). {0051] According to certain embodiments, the present invention provides anti-Tiez antibodies which interact with one or more amino acids tound within the ig‘t andlor lgE domains of Tie2.

The e(s} may consist of one or more contiguous sequences of 3 or more (eg, 3, 4, 5, 6, T, 8, 9, to, H, 12, 13, i4, 15, 16, 17, 18, 19, 20 or more} amino acids located within the Egg? and/or ng domains of TieZ. Alternatively, the epitope may consist of a piuraiity of noncontiguous amino acids (or amino acid sequences) located within the lg1 andlor ig2 s of Tie2. According to certain embodiments of the present invention, anti-Tie2 antibodies are provided which interact with one or more amino acids located within one or more amino acid segments selected from the group consisting of amino acids 536—105 of SEQ ID N07, amino acids 139-152 of SEQ lD NO:?; and amino acids 166—175 of SEQ iD N027. For example, the present invention inciudes ie2 antibodies which interact with at least one amino acid within each of the aforementioned segments (Le, within each of amino acids 96-108, 139-152, and 166—175 of SEQ lD N017). According to certain embodiments of the present invention, antibodies which interact with amino acids 139-152 and/or 166-175 of SEQ “3 N07 are capahie of biocking the ction between Tie2 and one or more Tie2 ligands, such as, e.g., Ang2 (see es 4-6, herein). {0052] Various techniques known to persons of ordinary skili in the art can be used to determine whether an antibody "interacts with one or more amino acids" within a ptide or protein. Exemplary techniques include, e.g, routine cross-blocking assay such as that described Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb, NY), alanine scanning mutational analysis, peptide blots is (Reineke. 2004. Methods Mol Biol 248:443—463), and peptide cleavage analysis. In addition, methods such as epitope excision, e extraction and chemical modification of ns can be ed (Tomer, 2000, n e 92487-496). Another method that can be used to identify the amino acids within a polypeptide with which an antibody interacts is hydrogenr’deuterium exchange detected by mass spectrometry. (See, 8.9., Example 4 herein). in general terms, the hydrogenfdeuterium exchange method involves deuterium-labeling the protein of interest, followed by binding the antibody to the deuterium-labeled n. Next, the protein/antibody complex is transferred to water to allow en-deuterium exchange to occur at all residues except for the residues protected by the dy (which remain deuterium-labeled). After dissociation of the antibody, the target protein is subjected to protease cleavage and mass ometry analysis, thereby revealing the deuterium—labeled es which correspond to the specific amino acids with which the antibody interacts. See, e.g., Ehring (1999) ical mistry 267(2):252—259; Engen and Smith (2001) Anal. Chem. 73:256A—265A.

The present invention includes anti-Tie2 antibodies that bind to the same epitope as any of the specific exempiary antibodies described herein leg, antibodies H1M2055N, and H2aM2760N, produced from deposited cell lines PTA-12295 and PTA-12296, respectively}.

Likewise, the present invention also includes anti—Tie2 antibodies that compete for binding to Tie2 or a TieZ fragment with any of the specific exemplary antibodies described herein (eg, antibodies Hill/2055M, and H2aM2?80N, produced from deposited cell lines PTA—12295 and PTA-12296, respectively). {0054] One can easily determine whether an antibody binds to the same epitope as, or competes for binding with, a reference anti—Tie2 antibody by using routine methods known in the art. For example, to determine if a test antibody binds to the same e as a reference anti-TieZ antibody of the ion, the reference antibody is allowed to bind to a Tie2 protein or peptide under saturating conditions. Next, the y of a test antibody to bind to the Tie2 molecule is assessed. If the test antibody is able to bind to Tie2 following saturation binding with the reference anti—TieZ antibody, it can be concluded that the test antibody binds to a different epitope than the reference ie2 antibody. On the other hand, if the test antibody is not able to bind to the Tie2 moiecule following saturation binding with the reference anti-Tie2 antibody, then the test antibody may bind to the same epitope as the epitope bound by the reference anti-Tie2 antibody of the ion. Additional routine experimentation (9.9., peptide mutation and binding analyses) can then be carried out to confirm whether the observed lack of binding of the test antibody is in fact due to binding to the same epitope as the reference antibody or if steric blocking (or another phenomenon) is responsible for the lack of observed binding. Experiments of this sort can be performed using ELiSA, RiA, Biacore, fiow cytornetry or any other quantitative or quaiitative antibody—binding assay avaiiabie in the art. In accordance with certain ments of the present invention. two antibodies bind to the same (or overlapping) e if, e. 9., a 1-. 5—, 10-, 20- or “EGO-fold excess of one antibody inhibits binding of the other by at ieast 50% but preferably 75%, 90% or even 99% as ed in a competitive binding assay (see, e.g., Junghans et ai.= Cancer Res. 19905014954502).

Aiternativeiy, two antibodies are deemed to bind to the same epitope if essentialiy ali amino acid mutations in the antigen that reduce or ate binding of one antibody reduce or eiiminate binding of the other. Two antibodies are deemed to have “overtapping epitopes" if oniy a subset of the amino acid mutations that reduce or eliminate binding of one antibody reduce or eiiminate binding of the other. {0055] To determine if an antibody competes for binding with a reference anti-Tie2 antibody, the above-described binding oiogy is performed in two orientations: in a first ation, the reference antibody is allowed to bind to a Tie2 le under saturating conditions ed by assessment of binding of the test antibody to the Tie2 moiecuie. in a second orientation, the test antibody is aiiowed to bind to a Tie2 molecule under saturating conditions foiiowed by assessment of binding of the reference antibody to the Tie2 moiecuie. If, in both orientations, only the first (saturating) antibody is e of binding to the Tie2 moiecuie, then it is conciuded that the test antibody and the reference antibody compete for binding to Tie2. As Witt be appreciated by a person of ordinary skit! in the art, an antibody that competes for binding with a nce antibody may not necessarily bind to the same epitope as the reference antibody, but may stericaiiy block binding of the reference antibody by binding an overiapping or adjacent epitope. ation of Human Antibodies {0056] Methods for generating onal antibodies, ing fuiiy human monocionai antibodies are known in the art. Any such known methods can be used in the context of the present ion to make human dies that specificatiy bind to human Tle2. {005?} Using VELD'CIivih’ltiixiETM technoiogy or any other known method for generating monocionai antibodies, high affinity chimeric antibodies to Tie? are initiaiiy isoiated having a human variabie region and a mouse constant region. As in the experimental section beiow, the antibodies are terized and seiected for desirabie characteristics, including affinity, seiectivity, epitope, etc. The mouse constant regions are replaced with a desired human constant region to generate the fuiiy human antibody of the invention, for exampie wiid-type or modified ith or igG4. Whiie the constant region seiected may vary according to specific use, high y antigen—binding and target specificity characteristics reside in the variabie region.

Bioequivalents {0058] The anti-Tie2 antibodies and antibody fragments of the present invention encompass ns having amino acid sequences that vary from those of the described antibodies but that retain the abiiity to bind human Tie2. Such variant antibodies and antibody fragments comprise one or more additions, deietions, or substitutions of amino acids when compared to parent sequence. but exhibit bioiogicai activity that is essentiaiiy equivalent to that of the described antibodies. Likewise, the anti-Tie2 antibody—encoding DNA ces of the present invention encompass ces that comprise one or more additions, deletions, or substitutions of nucieotides when compared to the disclosed sequence, but that encode an anti-Tie2 antibody or antibody fragment that is essentiaiiy bioequivalent to an anti-TieZ antibody or antibody fragment of the invention. {0059] Two antigen-binding ns, or antibodies, are considered bioequivaient if, for exampte, they are pharmaceutical equivaients or pharmaceutical alternatives whose rate and extent of absorption do not show a significant difference when administered at the same molar dose under r experimental conditions, either single does or muitiple dose. Some antibodies wilt be considered equivaients or pharmaceutical alternatives if they are equivaient in the extent of their absorption but not in their rate of absorption and yet may be considered bioequivaient because such differences in the rate of absorption are intentionai and are reflected in the labeling, are not essentiai to the attainment of effective body drug concentrations on, e.g., chronic use, and are ered medically insignificant for the particuiar drug product studied.

{D060} in one embodiment, two antigen—binding proteins are bioequivatent if there are no ciinicaity meaningful differences in their safety, purity, and potency. {0061] in one embodiment, two antigen-binding ns are bioequivaient if a patient can be switched one or more times between the reference product and the biotogical product t an expected increase in the risk of adverse effects, inciuding a clinicaily significant change in immunogenicity, or diminished effectiveness, as compared to continued therapy without such switching. {0062] in one embodiment, two antigen—binding proteins are bioequivaient if they both act by a common mechanism or mechanisms of action for the condition or conditions of use, to the extent that such mechanisms are known.

{D063} Bioeouivalence may be demonstrated by in vivo and in vitro methods. Bioequivalence measures include, e.g., (a) an in vivo test in humans or other mammais, in which the concentration of the antibody or its metabolites is measured in blood, piasma, serum, or other bioiogicai fiuid as a function of time; (b) an in vitro test that has been ated with and is abiy predictive of human in vivo bioavaiiability data; (c) an in vivo test in humans or other marnrnais in which the riate acute pharmacotogical effect of the antibody {or its target) is ed as a on of time; and (d) in a weil—controiied ai trial that establishes safety, efficacy, or bioavaiiability or bioequivalence of an antibody.

Bioequivalent variants of ie2 antibodies of the invention may be constructed by, _’§3_ for exampte, making s substitutions of residues or sequences or deieting terminai or internat residues or sequences not needed for biological activity. For example, ne residues not essentiai for bioiogicai activity can be deieted or replaced with other amino acids to prevent formation of unnecessary or incorrect intramolecular disutfide bridges upon renaturation, in other contexts, bioequivaient antibodies may include anti—Tie2 antibody variants comprising amino acid changes which modify the glycosylation characteristics of the antibodies, e.g., mutations which eliminate or remove glycosylation.

Species Seiectivity and Species Cross-Reactivity ing to certain embodiments of the invention, the anti—Tie2 dies bind to human Tie2 but not to Tie2 from other species. The present invention also inciudes anti-Tie2 antibodies that bind to human Tie2 and to Tie2 from one or more non—human species. For exempts, the anti-Tie2 antibodies of the invention may specificaily bind to human Tie2 as welt as to a rodent Tie2 (eg, Tie2 from mouse or rat). An exemplary construct that can be used to determine whether an antibody ically binds mouse Tie2 is the uct having the amino acid sequence of SEQ iD N08; an exempiary construct that can be used to determine Whether an dy specifically binds rat Tie2 is the uct having the amino acid sequence of SEQ iD NO:5. The use of these constructs to assess anti-Tie2 antibody binding is iilustrated in e 2, herein. tmmunoconjugates {0066] The invention encompasses anti—TieZ monoclonai antibodies conjugated to a therapeutic moiety ("immunoconiugate"), such as a cytotoxin, a chemotherapeutic drug, an immunosuppressant or a radioisotope. Cytotoxic agents include any agent that is detrimentai to ceils. Examples of suitable cytotoxic agents and chemotherapeutic agents for forming conjugates are known in the art, (see for exempts! W0 05/103081).

Muttispecific Antibodies The antibodies of the present invention may be monospecific, bi-specific, or pecific. Muitispecific antibodies may be specific for different epitopes of one target poiypeptide or may n antigen-binding domains specific for more than one target poiypeptide, See, e.g._. Tutt et at, 1991, .3. lmmunoi. i47:80—69; Kufer et at, 2004, Trends Biotechnot. 22:238-244. The anti—Tie2 antibodies of the present invention can be linked to or era-expressed with another functional moiecuie, e.g.. another peptide or protein. For e, an antibody or fragment thereof can be functionaiiy linked (e.g.. by chemical coupiing, genetic fusion, noncovaient association or otherwise) to one or more other moiecuiar entities. such as another antibody or antibody fragment to produce a bi-specific or a multispecific antibody with a second g specificity. For example, the present invention es bi-specific antibodies wherein one arm of an immunogiobulin is specific for human Tie2 or a fragment thereof, and the other arm of the giobuiin is specific for a second therapeutic target or is conjugated to a therapeutic moiety such as a trypsin inhibitor. {0068] An exemplary bi-specific antibody format that can be used in the context of the present invention invoives the use of a first giobuiin (lg) CH3 domain and a second ig CH3 domain, wherein the first and second ig CH3 domains differ from one another by at ieast one amino acid, and wherein at least one amino acid difference s binding of the bispecitic antibody to Protein A as compared to a bi-specitic antibody tacking the amino acid difference. in one embodiment, the first lg CH3 domain binds n A and the second lg CH3 domain contains a mutation that reduces or aboiishes Protein A binding such as an HQSR modification (by iMGT axon numbering; H435R by EU numbering}. The second CH3 may further comprise a YQBF modification (by iMGT; Y436F by EU). r modifications that may be found within the second CH3 inciude: D16E, LiSM, N448, K52N, VSTM, and V82i (by lMGT; DSSGE, L358M, N384S, K392N, VBQTM, and V422i by EU) in the case of tgGi antibodies; N448. KEEN, and V823 (iMGT; N384S, KSQZN, and V422i by EU) in the case of igGZ antibodies; and 015R, N448, KEEN, V57M, REQK, ETQQ, and V82! (by iMGT; Q355R, N3848, K392N, VSQTM, R409K, E419Q, and V422! by EU) in the case of lgG4 antibodies. Variations on the cific antibody format described above are contempiated within the scope of the present invention.

Therapeutic Formulation and Administration {0069] The invention provides pharmaceuticai compositions comprising the anti-Tie2 antibodies or antigen—binding fragments thereof of the present invention. The pharmaceuticai compositions of the invention are formuiated with ie carriers. excipients, and other agents that provide improved transfer, deiivery, toierance, and the like. A multitude of appropriate tormuiations can be found in the formulary known to ail pharmaceutical chemists: Remington‘s Pharmaceuticai Sciences, Mack Publishing Company, Easton, PA. These formuiations inciude, for s, powders, pastes. ointments, jellies, waxes, oiis, lipids, lipid (cationic or anionic) ning vesicies (such as LiPOFECTiNW), DNA conjugates, anhydrous absorption , oii-in-water and wa‘ter-in-oil emulsions, emulsions carbowax (poiyethyiene giycols of various rnoiecular weights}, semi—soiid gets, and semi-solid mixtures containing carbowax. See also Poweii et ai. “Compendium of excipients for parenteral forrnuiations“ PDA (1998) J Pharm Sci Technoi 52:238-311. {00M} The dose of dy administered to a patient may vary depending upon the age and the size of the patient, target disease, conditions, route of administration, and the iike. The preferred dose is typicaliy ated according to body weight or body surface area. When an dy of the present invention is used for treating a condition or disease associated with Tie2 activity in an aduit t, it may be advantageous to intravenously administer the dy of the present invention normaily at a singie dose of about 0.01 to about 20 mglkg body weight, more preferabiy about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg/kg body weight. Depending on the severity of the condition, the frequency and the duration of the treatment can be adjusted. Effective dosages and scheduies for administering Tie2 antibodies may be determined empirically; for exampie, patient ss can be red by periodic assessment, and the dose adjusted accordingly. Moreover, interspecies scaiing oi dosages can be performed using wail—known methods in the art {e.g., Mordenti et at, 1991, Phamtaceut.

Res. 8:13.51). {0071] Various delivery systems are known and can be used to ster the pharmaceuticai composition of the invention, e.g., encapsuiation in iiposomes, microparticies, microcapsuies, recombinant ceiis capabie of sing the mutant viruses. receptor mediated endocytosis (see, e.g., Wu et al., 1987, J. Bioi. Chem. 294432). s of introduction include, but are not iirnited to, ermai, intramuscular, intraperitoneai, intravenous, subcutaneous, intranasai. epidural, and orai routes. The composition may be administered by any convenient route, for exampie by infusion or bolus ion, by absorption through epitheliai or mucocutaneous linings (on, oral mucosa, rectal and intestinai mucosa, etc.) and may be administered er with other bioiogicaiiy active agents. Administration can be systemic or iocai. {0072] A pharmaceuticai composition of the present ion can be deiivered subcutaneously or intravenousiy with a standard needie and syringe. in addition, with respect to subcutaneous delivery, a pen delivery device readiiy has applications in delivering a pharmaceuticai composition of the t invention. Such a pen delivery device can be reusebie or disposable. A reusabie pen delivery device generaiiy utiiizes a repiaceabie cartridge that contains a pharmaceutical composition. Once aii of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readiiy be discarded and repiaced with a new cartridge that contains the pharmaceuticai composition. The pen delivery device can then be reused. In a disposable pen deiivery device, there is no repiaceable dge. , the disposabie pen ry device comes prefiiled with the pharmaceuticai composition heid in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical ition, the entire device is discarded. {0073] Numerous reusabie pen and autoinjector deiivery devices have appiications in the subcutaneous delivery of a pharmaceutical composition of the present invention. Exampies include, but are not iimited to AUTQPENTM (Owen Mumford, inc, Woodstock, UK), DiSE'i'RONiCTM pen (Disetronic Medicai Systems, Bergdorf, Switzerland), G MIX 75.125TM pen, HUMALOGTM pen, HUMALIN 7050“" pen (Eli Liily and Co, indianapoiis, IN), NOVQPENTM i, ii and iii (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNiDRTM (Novo Nordisk, agen, Denmark), EiDTM pen (Becton Dickinson, in Lakes, NJ), NTM, OPTiPEN PROM, OPTiPEN STARLETTM, and OPTICLlKTM (sahofi—aventis, Frankfurt, Germany), to name only a few. Exampies of disposable pen deiivery devices having appiications in subcutaneous deiivery of a pharmaceutical composition of the present invention e, but are not limited to the SCHJDSTARTM pen (sanofi—aventis), the FLEXPENTM (Novo Nordisk), and the iOi‘ii’li‘iF-‘ENTM (Eli Liily), the Stif-RECLiCl'tTM Autoinjector (Amgen, Thousand Oaks, CA}, the PENLETW (Haseimeier, Stuttgart, Germany), the EPiPEN (Day, LR), and the i-iLJh/llRATM Pen (Abbott Labs, Abbott Park EL}, to name oniy a few. in certain situations, the ceutical composition can be deiivered in a controiled reiease system. In one embodiment, a pump may be used (see Langer, supra; , 198?, CRC Crit. Ref. Biomed. Eng. 14:201). in another embodiment, polymeric materials can be used; see, Medical Applications of Controlled e, Langer and Wise (eds), 1974, CRC Pres, Boca Raton, Fiorida. In yet another embodiment, a controiied reiease system can be pieced in proximity of the composition’s target, thus requiring only a on of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controiied Reiease, supra, vol. 2, pp. 115-338). Other controlled release systems are discussed in the review by Langer, 1990, Science 249:1 527-1533. {0075] The injectabie preparations may e dosage forms for intravenous, subcutaneous, intracutaneous and uscuiar injections, drip infusions. etc. These injectabie preparations may be prepared by methods pubiiciy known. For example, the injectabie preparations may be prepared, e.g., by dissolving, suspending or emuisifying the antibody or its salt described above in a sterile aqueous medium or an oiiy medium conventionaliy used for injections. As the aqueous medium for injections, there are, for example, physiological saiine, an isotonic soiution containing giucose and other auxiiiary agents. etc., which may be used in combination with an appropriate iizing agent such as an aicohoi {e.g., ethanol), a polyaicohoi (e.g., propyiene giycoi, hyiene glycol), a nonionic surfactant [e.g., polysorbate 8C}, HUD—50 (poiyoxyethyiene (50 moi) adduct oi hydrogenated castor oii)}, etc. As the city medium, there are empioyed, e.g., sesame oii, soybean oil, etc., which may be used in combination with a soiubilizing agent such as benzyi benzoate, benzyi aicohoi, etc. The injection thus ed is preferabiy fitted in an appropriate ampouie. {0076] ageousiy, the pharmaceutical compositions for orai or parenterai use described above are prepared into dosage forms in a unit dose suited to fit a dose of the active ingredients. Such dosage forms in a unit dose inciude. for example, tabiets, piils, capsuies, injections (ampouies), itories, etc. The amount of the aid dy contained is generaiiy about 5 to about 500 mg per dosage form in a unit dose; aiiy in the form of injection, it is preferred that the aforesaid antibody is contained in about 5 to about 100 mg and in about 10 to about 250 mg for the other dosage forms.

Therapeutic Uses of the Antibodies {0077] The antibodies of the invention are useful, inter alia, for the treatment, prevention and/or ameiioration of any disease or disorder associated with Tie2 activity, inciuding diseases or disorders associated with angiogenesis. The dies and antigen—binding fragments of the present invention may be used to treat, e.g., primary and/or metastatic tumors arising in the brain and es, oropharynx, iung and bronchiai tree, gastrointestinai tract, mate and femaie reproductive tract. muscie, bone, skin and appendages, tive tissue, spieen, immune system, blood forming ceiis and bone marrow, iiver and urinary tract, and speciai sensory organs such as the eye. in certain embodiments, the dies and n—binding fragments of the invention are used to treat one or more of the ing cancers: renai ceii carcinoma, pancreatic carcinoma, breast cancer, prostate cancer, maiignant giiomas. osteosarcoma, colorectai cancer, maiignant mesotheiioma, multipie myeioma, ovarian cancer, smail ceii iung cancer, non-small ceii lung cancer, synoviai sarcoma, thyroid cancer, or Combination Therapies {D078} The present invention inciudes therapeutic administration regimens which comprise administering an anti-Tie2 antibody of the t invention in combination with at ieast one onai therapeuticaiiy active component. Non—iimiting exampies of such onai therapeuticaiiy active components include, for e, another Tie2 nist (e.g,, an anti— Tie2 antibody), an antagonist of epidermal growth factor or (EGFR) {e.g., anti-EGFR antibody [e.g., cetuxirnab or panitumumab} or smali moiecuie inhibitor of EGFR activity [e.g., geiitinib or eriotinibD, an antagonist of another EGFR family member such as rbB2, ErbBB or ErbB4 (eg, anti-ErbBZ, anti-ErbBB or anti-ErbB4 antibody or email molecuie inhibitor of Erbl32, ErbBS or ErbB4 activity), an antagonist of EGFRviii (9.9., an antibody that specificaiiy binds EGFRViii), a cMET anagonist (eg, an anti—GMET antibody), an IGF‘i R antagonist (e.g., an anti-iGF‘iR antibody), at B-raf inhibitor (e.g., yemurafenib, sorafenib, {BBC-0879, PLX—4720), a P‘DGFR-o tor tag, an anti-PDGFR-o antibody}, 3 PDGFR-fi inhibitor (e.g., an anti— PDGFR-fi antibody), 3 VEGF antagonist (e.g., a VEGF-Trap, see, 9.9., US 7,087,411 (aiso referred to herein as a "VEGF-inhibiting fusion protein"), anti—VEGF antibody (eg, bevacizumab), a smali moiecule kinase inhibitor of VEGF receptor (e.g., sunitinib, soratenib or pazopanib». a DLL4 antagonist (e.g.. an anti-DLL4 dy disclosed in US 2009f0142354 such as REGN42i ), an Ang2 antagonist (8.9., an anti-Ang2 antibody sed in US 2011i0027286 such as M H685P), etc. Other agents that may be beneficialiy administered in combination with the anti-Tie2 antibodies of the invention include cytoitine tors, inciuding smatl—molecuie cytokine inhibitors and antibodies that bind to cytokines such as iL-i, lL—2, Ii_-3, iL-4, IL—5, iL—6. iL-S, iL—Q, iL—ii, iL-‘i2. iL-13, lL—iT, iL-iB. or to their tive receptors. {0MB} The present invention also inciudes eutic combinations comprising any of the anti—Tie2 antibodies mentioned herein and an inhibitor of one or more of VEGF, AngZ, DLL4, ErbBZ, ErbB3, ErbB4, EGFRviii, cMet, lGFiR, B-raf, PDGFR—o, PDGFR—S, or any of the aforementioned cytokines, wherein the inhibitor is an aptamer, an nse moiecuie, a ribozyme, an siRNA, a ody. a nanobody or an antibody fragment (age, Fab fragment: F(ab')2 fragment; Fd fragment; FV fragment; scFv; dAb fragment; or other engineered moieoules, such as diabodies, triabodies, tetrabodies, minibodies and minimai recognition units). The anti—Tie2 antibodies of the invention may aiso be administered in combination with antivirais, antibiotics, anaigesics, osteroids andfor NSAIDs. The anti-Tie2’ antibodies of the invention may aiso be administered as part of a treatment regimen that step includes radiation treatment andfor conventionai chemotherapy. {0030] The additional therapeutically active componentis} may be administered just prior to, concurrent with, or shortiy after the administration of an anti-Tie2 antibody of the present invention; (for purposes of the t disciosure, such administration ns are considered the administration of an anti—Tie}.> antibody "in combination with" an additional therapeuticaiiy active component). The present invention inciudes pharmaceutical compositions in which an anti-TieZ antibody of the present invention is co—formuiated with one or more of the additionai therapeuticaiiy active componends) as described elsewhere herein.

Diagnostic Uses of the Antibodies {0081] The anti—Tie2 antibodies of the present invention may aiso be used to detect and/or measure Tie2 in a sampie. e.g.. for diagnostic purposes. For example, an anti-Tie2 antibody, or fragment thereof, may be used to diagnose a condition or disease terized by aberrant expression (9.9., over-expression, under—expression, tack of expression, etc.) of Tie2. ary stic assays for Tie2 may comprise, e.g., contacting a sampie, obtained from a patient, with an anti-TieZ antibody of the ion, wherein the anti—Tie2 antibody is labeled with a detectabie iabei or reporter molecule. Alternatively, an unlabeied anti-Tie2 antibody can be used in diagnostic appiications in combination with a secondary antibody which is itseif detectabiy labeied. The detectable iabei or reporter molecuie can be a radioisotope, such as 3H, ”C, 32F, 358. or 125i; a cent or chemiiuminescent moiety such as tluorescein isothiocyanate, or rhodamine; or an enzyme such as alkaline phosphatase, beta—gaiactosidase, horseradish peroxidase, or iuciterase. Specific exemplary assays that can be used to detect or measure Tie2 in a sampie inciude enzyme—iinked immunosorbent assay (ELiSA), mmunoassay (RIA), and fluorescence—activated ceii sorting (FACS).

Sampies that can be used in Tie2 diagnostic assays according to the present invention include any tissue or fiuid sample obtainable from a t which contains detectabie quantities of Tie2 protein, or fragments thereof, under normai or pathological ions. Generaiiy, leveis of Tie2 in a particular sample obtained from a y patient (9.9., a patient not afflicted with a disease or condition associated with abnormal Tie2 ieveis or activity) wiii be measured to initialiy estabiish a baseiine. or standard, ievel of Tie2. This baseiine level of Tie? can then be compared against the ieveis of Tie2 measured in samples ed from individuais suspected of having a Tie2 reiated e or condition.

EXAMPLES The foliowing exampies are put forth so as to provide those of ry skiil in the art with a compiete disclosure and ption of how to make and use the methods and compositions of the invention, and are not ed to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (eg, amounts, temperature, etc.) but some mentai errors and deviations shouid be accounted for. Unless indicated otherwise, parts are parts by weight, moiecuiar weight is e moiecuiar weight. temperature is in degrees rade. and pressure is at or near atmospheric.

Controt Constructs Used in the Following es An exemplary controi construct {anti—Tie? antibody) was included in l experiments described beiow for comparative purposes. The antibody, referred to as Control i, is a chimeric anti-Tie2 antibody with mouse heavy and tight chain variabie domains having the amino acid sequences of the corresponding domains of“12H8“, as set forth in US Patent 6,376,653. The constant domain of this antibody is human igG4.

Exampie 1. Generation of Human Antibodies to Human Tie2 Severai human anti—TieZ antibodies were generated by immunizing a VELOCIMMUNE® mouse with human Tie2 antigen according to standard methods (see. 9g, US Patent No. 6596541}. Using this technique, several anti-TieE antibodies were ed; exempiary dies generated in this manner . and their corresponding biological characteristics, are described in detail in the toliowing Exambies and inciude the antibodies designated H2aM2760N, H2aM27EtN, H1M2055N, Hit-£23048, H1H2317B, H1 H23228, H1H2324B. H1H23318, H1H2332B, l-i'i HZSSSS. Hi HZBBTB, H1 H23388, H1 H233QB, Ht H234OB, and H4H2055N. The H‘l M. HEM. Ht H, etc. prefixes on the antibody ations used herein indicate the particuiar Fc region at the antibody. For e. an "HEM" antibody has a mouse igG2 Fc. whereas an “H1 H" antibody has a human lgG‘i Fc. As wilt be appreciated by a person of ordinary skit! in the art, an Fc region of an antibody can be modified or repiaced with a different Fc region, but the variable domains {including the CDRs) wiii remain the same.

Hybridomas which produce the anti—Tie2 antibodies HtMEOSSN. and H23M2760N were deposited under terms in accordance with the Budapest Treaty with the American Type e Coiiection (ATCC), 10801 University Stud, as, Va. 20110—2209 on er 2, 201 1, under accession numbers PTA-12295 (H1M2055N) and PTA—12296 {H23MEYSON}.

Example 2. Surface Plasmon Resonance Derived g Affinities and Kinetic Constants of Human Monoclonal Anti-Tiez Antibodies Binding affinities and kinetic constants of human monoclonai ie2 antibodies were ined by surface piasmon resonance at 25°C and 3?°C (Tables 1-4). Measurements were conducted on a Biacore 2000 or T200 ment.

For antibodies with a mouse constant region (designated Hitvi or HEM), antibodies were immobiiized onto an anti-mouse Fc sensor surface and different concentrations of human, mouse or rat Tie2 constructs (hTieZ—His. mTieQ-hFc and rTieE—hFc} were injected over the antibody captured surface. For antibodies in the human 396 format (designated H‘iH or HéiH), either an anti-human Fc sensor surface (hTieZ-i—lis and ch) or anti-human Fab sensor surface {mTieZ-hFc and rTieZ-nFc) was ed depending on the Tie-2 construct applied to the antibody captured surface. Amino acid sequences of the constructs used in this e are as foitows: hTieZ-His (SEQ iD N052); 1052—1152553 ID N023); hTieZ—ch (SEQ tD N024); rTieZ—hFc (SEQ iD N05); and nFc {SEQ “3 N06}. {0039] Kinetic rate constants — association rate (ka) and dissociation rate {Rd} were determined by fitting the real time g sensorgrams to a 1:1 binding modei with mass ort limitation using Scrubber 2.0 curve fitting software. The equiiibrium dissociation constant (K13) and dissociative trait—fife (Ti/2) were calculated from the kinetic rate constants as: Kg {M} = kd I ka; and TV: (min) = (ln21(60*kd). ). As shown in Tables 1-2, severai antibodies demonstrated high affinity binding to hTieZ at both temperatures tested. in addition 5N, H1H23328. H1H23378, H1H234OB and H4H2055N ted icant binding to mouse and rat TieZ (Tables 34).

Table 1: Biaccre binding affinities of human mAbs to hTieZ at 25°C Binding at 25° Mab capture format Antibody its (1115“; Rd (5") K5 (Molar) TV: (min) nTieZ—His 2.91504 1.54504 5.32509 142501275014 nTie2-His 3.33504 1.01504 3.02509 15251112751 N ““5210 110524-115 5.75505 1.34504 2.33510 m H1 111205510 101924-115 1.99505 3 5.55509 _ H1 5 10152—ch 5.50505 3.75505 5.45511 nTieZ—His 1.43505 9.25504 5.50509 H1 1123175 51352ch 3.90505 2.35505 5.10511 nTie2—t-lis 1 57505 9.75504 5.54509 H1 1123223 hTieZ—ch 4.50505 1.39505 2.90511 5052—1-05 2.40505 9.25504 3.55509 H1 1123245 hTie2—ch 5.30505 5.92505 9.39511 5052—1415 3.53504 1.17503 3.22505 H1 H2331 e 10552—ch 5.20504 1 24504 1.52509 —05 4 2.23503 2.51505 1-11 H23325 11052—ch 1.25505 2.13504 1.1509 1123335 110524-115 5.92504 3.09504 5.21509 571524—115 5.47504 5.40504 1.00500 H1H23375 117102—ch 5 1.11504 9.30510 571524-115 4.93504 3.44504 0.99500 H1H23305 571152—1415 5.50504 1.25503 2.24500 _ 123305 117152—ch 1.51505 1.02504 5.70510 11711324115 7.01504 3.45503 4.02500 2340515 117102—ch_ 1.55505 1.01504 1.15500 _ 11711524113 3.82E+05 4.04504 1.27500 1441-1205514 117052—ch 1.27505 0.02505 7.80511 571024-115 5.53504 4 7.00500 0ontI'D11 117102—111 F0 1 45505 1.15504 8.03510 u NB = no binding under the conditions tested NT = not tested Table 2: Biacore binding affinities of human mAbs to hTieZ at 37°C Binding at 37° Mab e format ————— 112311215011 —————_:1:::;:_425+04_281704 _“::i“9 —————_0:::;:_‘01:“5_311:°4_31:7" ————— ————— ————— H‘HMB ————— H‘HWB ————— 1111123313 ————— ————— H‘H23388 ——1.935+05 2535504 1535-09 117521-135 9.75504 3 1 .02507 H1H234GB 07532—ch 2.00E+05 5.15504 2.55509 071524-113 5.495935 8.01504 1.45509 H4H2055N 11752—ch 1.57E+05 1.55504 9.30511 hTieZ-His 7.48504 1.12503 1.49508 00“ mt H 117152—ch 1.80505 1.70504 9.40510 NB = no g under the conditions tested NT = not tested Table 3: Biacore binding affinities of human mAbs to mTieZ at 25°C Binding at 25° Mab capture format NB = no binding under the ions tested Table 4: Biacore binding ties of human mAbs to rTieZ at 25°C Binding at 25°C Mab capture format H‘i H23403 rTieZ—hF0 1 43305 3.71 E-03 2.59505 H4H2055N rTie2-hFc i.07£+06 1.00500 9.31543 11550 NB = no g under the conditions tested Exampie 3. Epitope g ofTie‘Z mAbs using Luminex Beads {0090] To determine domain binding for the anti-Tie2 antibodies, severai h'i'ie2 receptor extracelluiar domain—deletion constructs were covalentiy linked to iuminex xMAP beads (Mpg/mi of each protein per 107 beads). The constructs are depicted in Figure i and are designated as toliows: hTie2 (lg‘l—ig2-E‘GF) {SEQ iD NO:7}; hTie2 (lg2—EGF} (SEQ ID NOS); h'i'ie2 {EGF} (SEQ ID NOE); nTieZ (Ig3-FN) (SEQ ID NO:10); and h'i'ieZ (Fix!) (SEQ ED N01“).

Aiso tested in this Example were engttr hTie2—ch (SEQ ID N014) and hTiei-hFc {SEQ ID N0212) ectodomain constructs. {0091] For binding, 25 pi of anti—Tie2 antibody {25 ug/mi) was added to 75 pi of the above created Luminex bead mixture (3x‘i i33 beads per construct) in binding buffer (PBS, 0.05% Tween 20, 1 mgfmi BSA, 0.05% sodium azide) into a 96 weli fiiter plate pore). Incubation was at room temperature (RT) for 90 min or overnight at 450 with shaking. The beads were washed 3x with washing buffer {PBS + 0.05% Tween 20) resuspended in 100 pl binding buffer containing PE (phycoerythrine)—Iabeled anti human kappa or PE—Iabeied anti mouse Fab secondary dy and incubated at RT for 45 min with shaking. Samples were washed 2X more and binding signal (MFI) for each bead was determined using a Luminex L200 or PBD instrument. Bead iinked human Tie2 was used as positive control and bead linked human Tiei was used to measure family cross-reactivity. in general MFE s greater than 500 units represent significant binding. Results are summarized in Table 5.

Table 5 hTieZ- hTiet h'l'iea hTie2 hTieZ hTie2 hriez AntibodV 2:32;?! ch -hFc (EGF) gz,ssi=) {Ig2.EGF) (FNi-hFc (warm-hes Domaig maintain 10205 i165? 8568 3403 14509 EGFJgs HiH2304B 4250 8901 8776 754a _—EGFrepeat —--n-———— —---————- —--n—n——— _--——_u—- ——-n————— ——--————n ——--————— ND = not determined {0092] From the above resuits, it can be conciuded that H2aM27’60N and H1M2055N bind to the igtz’igZ s of Tie2; HEaMZTSiN binds to the EGF and th domains of Tie2; H1H2304B, Hit-Q3178, H1H2322B and HiH2324B bind to the EGF domain of Tie2; and HtH233‘tB, H1H23328, H1H2337Ei, HtH233QB, Hit-£23408, and Controt I bind to the FN domain of Tie2.

Exampte 4. Epitope Mapping of H4H2055N Binding to Tit-22 by HID Exchange {0093] Experiments were conducted to more preciseiy define the amino acid residues of Tie2 with which H4H2055N interacts. {H4H2055N is a futly human igG4 version of the antibody H1M2055N produced from hybridoma 295.) For this purpose HID exchange epitope mapping was carried out. A general description at the HID ge method is set forth in 6.9., Ehring (1999) AnaMicaf Biochemistry 267(2):252—259; and Engen and Smith (2001)Anaf.

Chem. 73:256A-265A. {0094] To map the binding et's) of antibody H4H2055N on Tie2 Via H/D exchange, a inant construct consisting of the igt, ig2 and EGF extraceiiuiar domains of human Tie2 (hTieZ (igt-igZ—EGF); SEQ ID Nil?) was used. Antibody H4H2055N was covalentty attached to N—hydroxysuccinimide (Ni-i8) agarose beads (GE Litescience). {0095] in the 'on-soiutionlott—beads‘ experiment (on—exchange in solution foiiowed by ott— exchange on beads), the iigand {hTieZ ig’i-IgZ—EGF) was deuterated for 5 min or ’30 min in PBS buffer prepared with D20, and then bound to 5N beads through a 2 min incubation. The ound beads were washed with PBS aqueous butter (prepared with H20} and incubated for hait of the tin-exchange time in PBS buffer. After the off—exchange, the bound Tie2 was etuted from beads with an ice-cold tow pt-I TFA soiution. The eluted Tie2 was then digested with immobitized pepsin {Thermo Scientific) for 5 min. The resulting es were desatted using ZipTip® chromatographic e tips and immediately anaiyzed by teXtreme matrix assisted iaser desorption ionization time of flight (MALDI-TOF) mass spectrometry (MS). {0096] in the ’on-beadsioff—beads' experiment change on beads totiowed by off- exchange on beads). Tie2 was first bound to H4H2055N beads and then incubated for 5 min or ‘30 min in D20 for tin—exchange. The foliowing steps (oft-exchange. pepsin digestion, and MS anatysis) were carried out as described for the 'on-soiution/off-beads' procedure. The centroid vaiues or average o—charge ratios (miz) of at! the detected peptides were ated and compared between these two sets of experiments. {0097] The resuits are summarized in Table 5 which provides a comparison of the centroid miz values for sit the ed peptides identitied by tiquid chromatography—matrix assisted iaser desorption ionization (LC-MALI)” MS foiiowing the H.113 exchange and peptic digest procedure.

Likely due to disuifide bonds, the Tie2 sequence coverage detected from a singie MALDLTOF um is reietiveiy tow. Nevertneiess. mere than haif of the detected peptic peptides gave simiier id vaiues for both the on-soiutionfoff—besds and on-beads/off—beeds protocols.

Three segments with corresponding residues 88-106, 139-152, and 166-175 had deita centroid vaiues 2 0.20 mi: in both experiments. For purposes of the present Exampie a positive difference (A) of at ieast 0.20 mi: in both experiments indicates amino acids protected by dy g. Segments meeting this criterion are indicated by boid text and an asterisk (*) in Tebie 6.

Table 6 : H4H2055N Binding to Tie2 Experiment 1 ment II min on-I 2.5 min change 10 min on-I 5 min off-exchange Residues {of SEQ iD Uri-solution On-Beads I On-soiution N07) 1 Off Beads Off-Beads A I Off Beads ——-m 42-53 1440.52 0.08 1440.82 1440.52 0.10 4258 2041.43 0.01 2041.58 2041.41 0.17 7887 1124.17 0.03 1124.20 1124.14 0.07 8885 1048.48 0.02 1048.48 1048.54 0.08 1 1787.78 0.27 1787.78 1787.57 33-10? 1881.08 1881.14 1330.88 931-101“ 1127.80 1127.80 1127.81 0.19 83-1021 124148 1241.34 1240.88 0.51 era-102* 850.54 850.55 850.25 SIS-106* 1233.28 1233.43 1232.85 0.78 139451" 1514.22 1.08 1514.48 151321 1.28 1394.52" 1843.17 1.16 1843.44 1842.08 1.38 152—185 1488.87 0.14 1488.88 1488.77 0.11 152—188 1882.08 0.07 1882.10 3 0.02 158—185 1388.70 0.08 7 1888.88 0.11 188-175* 1115.07 0.47 1115.04 1114.85 0.39 167475" 951.73 951.30 951.74 951.33 0.41 {0098] Since the peptide fragment (mlz of 1049.4} corresponding to residues 88-95 did not show deuteron retention (£1 = 0} after off-exchange, the 1First ted t (88-106) can be reduced to oniy inciude es 96-106. Thus the three regions 90-106. 139-152, and 100-175 of SEQ iD NO:7 are protected from fuii oft-exchange by H4H2055N binding to Tie2 after cn- exchange. {0099] The 191 domain of Tie2 ts of amino acids 1-97 of SEQ iD NO:7; the 192 domain of Tie 2 consists of amino acids 98-186 of SEQ 10 N07; and the EGF repeat domain consists of amino acids 187—321 of SEQ iD N07. Thus the tirst binding region (amino acids 96—306 of SEQ iD NO?) includes the East two amino acids of the tg’i domain and the first 9 amino acids of the ig2 domain, The second and third g regions (amino acids 139-152 and 166-175 of SEQ iD NO?) are iocated ty Within the igZ domain. it should also be noted that the second and third binding regions (amino acids 139-352 and 166-175 of SEQ lD N017) tie within the amino acid region of Tie 2 that iy contacts its cognate iigand, Angiopoietin-Z, based on the co-crystal structure by Barton et at. (2006, Nat Struct Moi Biol 13(6) 524—532}. Therefore, this example suggests that antibody H4H2055N binds a discontinuous epitope ily within the ig2 domain of human Tie2 (with one or two potentiai amino acid contacts within the C-terminal portion of the £91 domain), and that binding to these regions correiates with the abiiity 0t H4H2055N to block the interaction between Tie2 and Ang2 as rated in Examples 5 and 5 herein.

Exampie 5. Assessment of the Abiiity of Anti-Tie2 Antibodies to Block the Interaction Between Tie2 and Angiopoietins {0101] Tie2 is known to interact with angiopoietins (Ang‘i, Ang2, Ang3 and Ang4). An initiai set of ments was ore conducted to evaiuate the abiiity of anti-TieZ antibodies to biock Tie2 binding to Ang2. in this tirst set of experiments, a quantitative biocking immunoassay was utiiized. Briefly, solutions of 0.8 nM biotinyiated ecto—Tie2 tused to SxHis (SEQ iD N02) were premixed with anti—TieZ antibody g from ~ 50 nM to 0 nM in serial diiutions. After a 1-hour incubation at room temperature, the amount of Tie2—His bound to plate coated Bow- Ang2 was ed by sandwich ELISA. Bow-AngZ (SEQ ED N0113}, an Ang2 construct that comprises the Fc domain of hith flanked by one FD domain ct Ang2 at both termini, was coated at 2 ugfmi on a 96 watt microtiter plate and biocked with BSA. Plate—bound biotin-TieZ- His was detected using HRP conjugated streptavidin, and developed using BD OotEIATM (BD Biosciences Pharmingen, San Diego, CA). Signals of 0045;) nm were recorded and data was anaiyzed using GraphPad Prism to ate iCSG values. Results are summarized in Tobie 7. ings were d as the amount of dy required to reduce 50% of biotin-Tie2—His detectabie to piste bound Bow-AngE iigand. in Tabie 7‘ an antibody is designated an “enhancer" if the addition of the antibody increases the amount of Tie2-His bound to the Bow- Ang2—coated sun‘ace relative to the no antibody control.

Table 7: lC5g values for anti-Tie?! Mabs blocking of hTie2 to plate bound Bow-AngZ Result in Biocking ot binding Stocking to Bow—AngZ d ELlSA [C53 M H23M2760N HZaMZTBi N Enhancer Nz’A Hi MZDSSN Enhancer N/A Ht H2304B Enhancer MA H1H2317B Enhancer N/A H1H23228 Enhanfier H1H2324B Enhancer Ht H2331 B Enhancer H1H2332B Enhancer H‘i HZSBBS Enhancer H1H2337B Enhancer Ht HZSSSB er H1H23398 r H1H234OB Biocker H4H2055N Biocker Controi l Enhancer {0102] This first set of experiments demonstrates that anti-Tie2 antibodies HiHESSQB, H1H234DB and H4H2055N are abie to block the interaction between AngZ and Tie2 in an ELiSA format {0103] A further set of experiments was conducted to evaluate the ability of H4H2055N to biock Tie2 binding to AngZ and other members of the angiopoietin famiiy (Angt, Angs and Ang4). in this set of experiments, an Octet Red sor was employed using two experimentai formats. in the first format, hTieZch {SEQ lD NO:4: it} pglml) or a negative i was captured on anti—ch Octet senor tips for 5min. The captured sensor tip surfaces were then saturated with H4H2055N or Control l antibody by dipping into wells containing 300 nM of tive mono‘cional antibodies for 10 min. Finally the sensor tips were placed into welis containing tOOnM of hAngt {R&D Systems, inc, Minneapolis, MN, Cat 923—AN/CF; Accession #QSHYAO), hAngZ (R&D Systems, inc, polis, MN, Cat 623-AN/CF; Accession #015123), mAngS (R80 Systems, inc, Minneapolis. MN, Cat 738-ANXCF; Accession #QQWVHB) or hAng4 (R&D Systems, lnc., Minneapolis, MN, Cat 964-AN/CF; Accession Q9Y264) for 5 min. Binding response at each step of the experiment was monitored and the binding of ent oietins to the monoctonal antibody saturated receptor surfaces was piotted (Figure 2). As illustrated by Figure 2, H4H2055N biocked the binding of all four angiopoietins to the hTie2.ch sun‘ace. The Control i antibody did not biock binding of any of the angiopoietins to the coated surfaces. in the second assay format, hTieZch (10‘ gig/mi) or negative control was captured on anti—ch coated Octet sensor tips (5 min), ed by placing the tips into ent weiis containing 100‘ anl of angiopoietin , hAng2, mAngB or hAngii) for 1C) min. Lastly, the senor tips were placed into welis containing 300nM of H4H2055N or Control i (5min). As in format 1, the binding response at each step was monitored and the binding response was piotted (Figure 3). This format demonstrated that, unlike Controi l, the binding of H4H2055N is diminished by the aiready bound angiopoietins on the Tie2-coated surfaces. Thus, H4H2055N competes with the oietins (Angt, AngZ, AngS and Ang4) for binding to Tie2.

Exampie 6. Assessment of the Abiiity of Anti-Tiez Antibodies to Biock Angiopoietin- Mediated Tie2 ing To further characterize anti-hTieZ mAbs of the invention. the abiiity of the antibodies to biock Tie2-induced inciterase activity was ed. Briefly, HEK293 cells were stabiy transfected with hTie2, and the top 10% of Tie2 expressing ceiis were isoiated via FACS.

These coils were then transduced with a serum response element (SRE}-dependent iuciterase reporter ientivirus (SA Biosciences), and a cionai popuiation {293—TiE2iSRE—Luc) with robust response to tetran'ieric forms of angiopoietin—i (Bow-Angi, SEQ ii} NO:i4) and oietin-Z (Bow—AngZ, SEQ ED NCMB) was isoiated. {0106] For assays, 293—Tie2/SRE-Luc ceiis were seeded (30,000 cells per well) in EBB—weii plates one day prior to ent. Dose-response curves were ted with serial ditutions of Bow-Angi (8A1) or Bow—Ang2 (8A2) incubated with ceiis for 411 before measurement of iuciferase activity. For Tie2 tion, ceiis were d with ly diiuted Tie2 mAbs in the ce of a 1 11M constant dose of BM or 8A2 for 4 ii. Luciferese activity was measured on a Victor turninometer toiiowing incubation with Cine-Git)TM (Promege) reagent. Three ciasses of antibodies were observed: Blockers, defined as a reduction in Tie2 dependent iuciferase activity upon addition of antibody; Activators. defined as an increase in TieZ dependent luciferase activity upon addition of antibody; and Not Active. defined as no significant change in signai upon their addition. For “Not Active" antibodies, no EC5Q or H350 value was calcuiated. Results are summarized in Tabie 8.

Tame 8: tcsn values for Anti-Tie: antibodies E653 or |C5o i SEM ECfifi Oi' K350 i SEM Main mass (10'? M) Maia Ciass (10-9 M) Antibod 'BowAn’i BowAn I i . BowAn2 60N 11.6 a .81 Blocker 2.8 i .14 Beige ‘0 H1H2324B No activity 1-111—123315 Activator 13.6 i 3.1 H1 stsze Activator 2.5 i 1.2 3.? i- .15 H1 H23338 No activity NA NA Hit-£23378 Activator 1.1 i 02 4.7 i .80 H1H2338U3 No activity NA NA 1111-1233193 Activator me i .23 HiH234cie Activator 1.4i i 01 Control 1 mm vaiues are reported for Biockers, and E650 vatues are reported for Activators {0107] Anti—TieE antibodies HZaMEYBON, HEaMZYE’iN and H4H2055N were identified as biockers in this experimental system.

Exampte 7. ment of the Anti-Tumor Activity of an Anti-Tie? Antibody in viva {0108] An exemplary anti—Tiez antibody 55N) was tested for its abiiity to inhibit the growth of human tumor xenografts in immunocompromised mice. Briefly, either human coiorectal HT29 or Coio205 tumors were grown in SCiD mice. When tumors were paipabie (100 mm3 for HTZQ and 400mm3 for Coio205} animais were d biweekly with Fe protein (“i O mgiitg) or anti-TieZ antibody (H1M2055N; 10 mgfkg). At the end of treatment (31 days for HT29 & 14 days for CoioEOS), the percent tumor growth tion (%TGi} was determined (Tabie 9} and turner tissue was harvested and utiiized tor vessei density analysis. Vascular density was assessed in 30pm thick OCT tumor sections by 0031 immunohistoohemistry. Nit-i image software was utiiized to ine the % area vessel density in the tumor tissue ns.

Resuits are iiiustrated cally in Figures 4—7.

Table 9: Percent Decrease in Tumor Growth with Anti-TieZ Maia Treatment Avg Tumor Avg Tumor Growth (mmS) % Decrease in Growth {mmS} % se in dy from start of Tumor Growth from start of Tumor Growth treatment treatment Fe n H2M2055N {0109] As shown in this Exampie, the anti-Tie2 Ab, H3M2055N, not oniy decreased tumor growth (Tabie 9), but aiso significantly decreased vessei density in both HTZQ (25%. Figure 5) and Col020‘5 (40%, Figure 7) xenograft models.

The present invention is not to be iimited in scope by the specific embodiments describe herein. indeed. various modifications of the invention in addition to those described herein Witt become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fail within the scope of the appended ciaims.

BUDAPEST RESTRICTED ICATE OF DEPOSIT BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE ATIONAL FORM RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT ISSUED PURSUANT TO RULE 7.3 AND VIABILITY ENT ISSUED PURSUANT TO RULE 10.2 The an Type Culture Collection (ATCC®) has received your deposit of seeds/strain(s)/strain(s) in connection with the filing of an application for . The following ation is provided to fulfill Patent Office requirements.

Dr. Nicholas Papadopoulos Regeneron Pharmaceuticals 777 Old Saw Mill River Road Tarrytown, NY 10591-6707 Deposited on Behalf of: Re eneron Pharmaceuticals Date of Receipt of seeds/strain(s) by the ATCC®: December 02I 2011 identification Reference b De ositor: ATCC ®Patent De osit Desi nation: Quantity Received: Hybridoma isolated from mouse spleen: VLR07A05-2 PTA-12295 25 vials Hybridoma isolated from mouse spleen: FAKU1OB10 PTA-12296 25 vials The ATCC® understands that: 1. The deposit of these seeds/strain(s) does not grant ATCC® a license, either express or implied, to ge the patent, and our release of these seeds/strain(s) to others does not grant them a license, either express or d, to infringe the patent. 2. If the deposit should die or be destroyed during the effective term of the patent, it shall be your responsibility to replace it with viable material. It is also your responsibility to supply a sufficient ty for bution for the deposit term. ATCC® will distribute and maintain the material for 30 years or 5 years following the most recent request for the deposit, whichever is longer. The United States and many other countries are signatory to the Budapest Treaty.

Prior to the issuance of a US. Patent, the ATCC® agrees in consideration for a one-time service charge, not to distribute these seeds/strain(s) or any ation relating thereto or to their deposit except as instructed by the depositor or relevant patent office. After nt patent issues we are responSIble to release the seeds/strain(s) and they Will be made available for distribution to the public without any restrictions. We will inform you of requests for the seeds/strain(s) for 30 years from date of deposit. -32a- The deposit was tested December 22I 2011 and on that date, the seeds/strain(s) were viable Internationat Depository Authority: American Type e Collection (ATCC®), Manassas, VA, USA Signature of person having authority to represent ATCC®: —————— December 28I 201 1 ATCC® Patent Depository Date cc: Valeta Gregg-Emery. Ph.D. J.D.

Ref: Docket or Case No: 7100A -32b- THE

Claims (10)

CLAIMS DEFINING THE ION ARE As FOLLOWS
1. An ed antibody or antigen-binding fragment thereof that specifically binds human Tie2 and blocks the interaction between Tie2 and all four of An 91, An 92, Ang3 and An 94, wherein the antibody or antigen-binding fragment interacts with amino acids 96.06 of SEQ ID N0:7, amino acids 139-152 of SEQ ID N0:7; and amino acids 166.75 of SEQ ID N0:7, as determined by hydrogen/deuterium eXchange, and comprises the heavy and light chain complementarity determining regions (CDRs) of the antibody produced from a cell line deposited with the American Type Culture Collection under accession number PTA-12295
2. 2. The isolated antibody or antigen-binding fragment of claim I, wherein the antibody or antigen-binding fragment f specifically binds a rodent Tie2 and human Tie2
3. 3. The isolated antibody or antigen-binding fragment of claim 2, wherein the rodent Tie2 is mouse Tie2 or rat Tie2
4. 4. The isolated antibody or antigen-binding fragment of claim I, wherein the antibody or n-binding fragment thereof specifically binds human, mouse and rat Tie2
5. 5. An isolated antibody or n-binding fragment thereof produced from a cell line deposited with the American Type e Collection under accession number PTA- 12295
6. 6. An isolated antibody or antigen-binding fragment thereof produced from a cell line ted with the American Type e Collection under accession number PTA- 12296
7. 7. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims I to 6 and a pharmaceutical Iy acceptable carrier or diluent
8. 8. A pharmaceutical composition for use in inhibiting the growih of a tumor in a patient, the pharmaceutical composition comprising an dy or antigen-binding fragment thereof of any one of claims I to 6, and a ceutical Iy acceptable carrier or diluent
9. 9. Use of an antibody or antigen-binding fragment f of any one of claims I to 6 in the cture of a medicament for inhibiting the growth of a tumor in a patient
10. 10. The antibody or antigen binding fragment according to any one of claims I to 6, the composition according to claim 7 or 8, or the use according to claim 9, substantially as hereinbefore described REGENERON PHARMACEUTICALS, INC WATERMARK PATENT AND TRADE MARKS ATTORNEYS P38476NZOO